Peptides specific for Mycobacterium avium subspecies paratuberculosis infection: diagnostic potential.

Mycobacterium avium subspecies paratuberculosis (Map) is the causative agent of Johne's disease (JD). Current serological diagnostic tests for JD are limited by their sensitivity when used in sub-clinical stages of the disease. Our objective was to identify peptides that mimic diagnostically important Map epitopes that might be incorporated into a new-generation JD diagnostic. Four peptides were isolated from a phage-displayed random peptide library by screening on antibodies derived from Map-infected goats. The peptides were recognised by antibodies from Map-infected goats but not by antibodies from uninfected goats. The peptides elicited immune responses in rabbits, which reacted strongly with bona fide Map antigens proving the peptides were true epitope mimics. To assess the diagnostic value a panel of goat sera was screened for reactivity's with peptides. The peptides were recognised by antibodies from a proportion of goats infected with Map compared with control animals with a diagnostic specificity of 100% and the sensitivity ranged from 50 to 75%. Combinations of any two peptides improved sensitivity 62.5-87.5% and 100% sensitivity was achieved with three of the four peptides in combination. These data suggest peptides representing diagnostically important Map epitopes could be incorporated into a sensitive diagnostic test.

A long-term bacteriological and immunological study in Holstein-Friesian cattle experimentally infected with Mycobacterium avium subsp. paratuberculosis and necropsy culture results for Holstein-Friesian cattle, Merino sheep and Angora goats.

The aims were to longitudinally evaluate the interferon-gamma (IFN-gamma) test in comparison to faecal culture and the absorbed ELISA in a cattle infection model for Johne's disease and to determine the adult infection status, by necropsy and tissue culture, of sheep, goats and cattle infected as young animals. Clinical disease, faecal culture results and immunological responses for Merino sheep [Stewart, D.J., Vaughan, J.A., Stiles, P.L., Noske, P.J., Tizard, M.L.V., Prowse, S.J., Michalski, W.P., Butler, K.L., Jones, S.L., 2004. A long-term study in Merino sheep experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies. Vet. Microbiol. 104, 165-178] and Angora goats [Stewart, D.J., Vaughan, J.A., Stiles, P.L., Noske, P.J., Tizard, M.L.V., Prowse, S.J., Michalski, W.P., Butler, K.L., Jones, S.L., 2006. A long-term study in Angora goats experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies. Vet. Microbiol. 113, 13-24], in the same experiments as the Holstein-Friesian cattle, have been described. Two longitudinal experiments involving Holstein-Friesian cattle challenged with either bovine or ovine strains of Mycobacterium avium subsp. paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the IFN-gamma test and the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Cell-mediated (CMI) responses were substantially higher for the bovine Map strain during the 42-month period following dosing but then declined in the remaining 12 months. However, for the ovine Map challenge and control groups, CMI responses were not significantly different from each other. None of the cattle developed clinical disease and only one of the cattle in the bovine Map gut mucosal tissue challenged group was a persistent faecal shedder and also an ELISA antibody responder which developed after shedding commenced. Culture of tissues, following necropsy at the completion of the experiments, showed no evidence of infection in any of the challenged cattle and sheep for either the bovine or ovine Map strain in contrast to positive cultures for challenged goats in the same experiments. The tissues from the control cattle, sheep and goats were culture negative. The cattle were less susceptible to the bovine and ovine Map strains than goats and sheep with the goats being the least naturally resistant.

A long-term study in Angora goats experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies.

Two longitudinal experiments involving Angora goats challenged with either bovine or ovine strains of Mycobacterium avium subspecies paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the interferon-gamma (IFN-gamma) test and the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Persistent shedding, IFN-gamma production, seroconversion and clinical disease occurred earlier with the bovine Map gut mucosal tissue challenge inoculum than with cultured bacteria. The IFN-gamma responses of the gut mucosal tissue and bacterial challenge groups were substantially and consistently higher than those of the control group. The in vivo and cultured cattle strains were much more pathogenic for goats than the sheep strains with persistent faecal shedding, seroconversion and clinical disease occurring in the majority of bovine Map challenged goats. With the ovine Map, 3 goats developed persistent antibody responses but only one of these goats developed persistent faecal shedding and clinical disease. However, there was no significant difference between the IFN-gamma responses of the tissue challenged, bacterial challenged and control groups. Compared with sheep, the ELISA appeared to have higher sensitivity and the IFN-gamma test lower specificity.

Development of a Johne's disease infection model in laboratory rabbits following oral administration of Mycobacterium avium subspecies paratuberculosis.

To assess the rabbit as a model for the study of Johne's disease pathogenesis, a breeding group of adult and juvenile New Zealand white rabbits were orally challenged with three doses of the Mycobacterium avium subspecies paratuberculosis wildtype bovine strain, CLIJ623, on three occasions. Faecal culture, post-mortem tissue bacteriological culture and histopathology were used to monitor the disease progression in the rabbits for more than 2 years. Of 4 adult and 16 juvenile orally dosed rabbits M. paratuberculosis organisms were recovered bacteriologically from two and three animals, respectively, using the BACTECtrade mark radiometric culture system. Tissue sites from which the bacteria were recovered included the mesenteric lymph nodes, ileocaecal valve, vermiform appendix, caecum, proximal colon and jejunum. Body weight loss, reduced abdominal fat and mild lesions were observed at necropsy in four infected rabbits. Diarrhoea and persistent faecal shedding of bacteria were not observed. Faecal culture did not yield any cultivable mycobacterial organisms on solid media.

A long-term study in Merino sheep experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies.

Two longitudinal experiments involving Merino sheep challenged with either bovine or ovine strains of Mycobacterium avium subsp. paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the interferon-gamma test, the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Infections were induced with either a bovine or ovine strain of Map in separate experiments with infections being more easily established, in terms of faecal bacterial shedding and clinical disease when the challenge inoculum was prepared from gut mucosal tissue than cultured bacteria. The patterns of response for shedding and clinical disease were similar. Cell-mediated immune responses were proportionally elevated by at least an order of magnitude in all sheep dosed with either a bovine or ovine strain of Map. Conversely, antibody responses were only elevated in a relatively small proportion of infected sheep. Neither of the clinically affected tissue challenged sheep developed an antibody response despite the presence of persistent shedding and the development and decline in cell-mediated immunity. The results indicated that for sheep the interferon-gamma test may be useful for determining if a flock has been exposed to ovine Johne's disease.

Yield of colonoscopy in patients with nonacute rectal bleeding: a multicenter database study of 1766 patients.

OBJECTIVE: There are few data to guide the choice between colonoscopy and flexible sigmoidoscopy in patients with nonacute rectal bleeding, especially in younger age groups. Our aim was to determine the yield of colonoscopy for significant proximal large bowel disease in the absence of significant distal disease, with special reference to young patients. METHODS: This was a retrospective study of data collected prospectively in 1766 patients (median age 57 yr, 711 women). The endoscopic database (GI-Trac) contained 152 discrete fields for data input. Multiple logistic regression analysis was performed to identify variables independently associated with the presence of isolated significant proximal disease. RESULTS: Young patients had a higher percentage of normal examinations than did older patients. The incidence of diverticular disease, small polyps, large polyps, and cancer rose with increasing age. No patient aged <40 yr had an isolated proximal cancer, but 7% had other significant isolated proximal disease. There was no overall association between age and significant proximal disease in the absence of significant distal disease (p = 0.66). The only variable associated with isolated proximal disease was anemia (odds ratio = 1.81; 95% CI = 1.11-2.93; p = 0.02). CONCLUSION: The yield of colonoscopy (beyond the range of sigmoidoscopy) for neoplasia is low in patients aged <40 yr, but other significant disease may be missed if age is the only criterion determining colonoscopy use.

Anti-mosquito midgut antibodies block development of Plasmodium falciparum and Plasmodium vivax in multiple species of Anopheles mosquitoes and reduce vector fecundity and survivorship.

The mosquito midgut plays a central role in the sporogonic development of malaria parasites. We have found that polyclonal sera, produced against mosquito midguts, blocked the passage of Plasmodium falciparum ookinetes across the midgut, leading to a significant reduction of infections in mosquitoes. Anti-midgut mAbs were produced that display broad-spectrum activity, blocking parasite development of both P. falciparum and Plasmodium vivax parasites in five different species of mosquitoes. In addition to their parasite transmission-blocking activity, these mAbs also reduced mosquito survivorship and fecundity. These results reveal that mosquito midgut-based antibodies have the potential to reduce malaria transmission in a synergistic manner by lowering both vector competence, through transmission-blocking effects on parasite development, and vector abundance, by decreasing mosquito survivorship and egg laying capacity. Because the intervention can block transmission of different malaria parasite species in various species of mosquitoes, vaccines against such midgut receptors may block malaria transmission worldwide.

Brugia malayi microfilariae (Nematoda: Filaridae) enhance the infectivity of Venezuelan equine encephalitis virus to Aedes mosquitoes (Diptera: Culicidae).

We examined the potentially conflicting effects that microfilarial (MF) enhancement of viral infectivity and MF-induced mortality in mosquitoes have on the vectorial capacity of Aedes aegypti (L.), Aedes triseriatus (Say), and Aedes taeniorhynchus (Wiedemann) for Venezuelan equine encephalitis virus (VEE) when mosquitoes feed on gerbils co-infected with Brugia malayi (Buckley). Groups of mosquitoes were fed on gerbils that were either dually infected (VEE plus B. malayi MF) or singly infected (VEE only). Mosquito mortality was recorded daily, and 5-8 d later, surviving mosquitoes were assayed for disseminated viral infection. The contrasting effects of MF enhancement and MF-induced mortality differed among mosquito species and were determined by the nature and consequences of MF penetration through the mosquito midgut, but not to differences in mosquito susceptibilities to parenterally introduced virus. In Ae. aegypti, MF-induced mortality was high and tended to eliminate any significant effect of MF enhancement. In Ae. triseriatus, MF-induced mortality was low, and feeding on dually infected hosts resulted in 9 times as many mosquitoes with disseminated viral infections as did feeding on singly-infected hosts. In Ae. taeniorhynchus, MF-induced mortality was extremely high, yet under our experimental conditions, feeding on a dually infected hosts resulted in nearly 30 times as many disseminated infections as did feeding on singly infected hosts. The final outcome on vectorial capacity depended on the specific combination of MF, virus, and mosquito species involved. Therefore, future efforts toward understanding MF enhancement should be directed toward mosquito-virus-parasite species combinations that occur together in nature.

Infectivity of Plasmodium berghei sporozoites delivered by intravenous inoculation versus mosquito bite: implications for sporozoite vaccine trials.

Plasmodium berghei sporozoites delivered by mosquito bite were more infectious to outbred CD-1 mice than were sporozoites delivered by intravenous inoculation. The route of challenge also affected vaccine efficacy. In view of these findings and the fact that mosquito bites are the natural mode of sporozoite delivery, infectious mosquito bites should be considered the challenge protocol of choice for sporozoite vaccine efficacy trials.

Quantitation of cat immunoglobulins in the hemolymph of cat fleas (Siphonaptera: Pulicidae) after feeding on blood.

Passage of ingested cat immunoglobulin G (IgG) into the hemocoel of cat fleas, Ctenocephalides felis (Bouché), was examined using antibody capture enzyme-linked immunosorbent assays (ELISA) and Western blotting. Fleas were fed heparinized cat blood via membrane feeders. Cat IgG was present in the hemolymph of engorged female fleas 1 h after ingestion at an estimated quantity of 35 +/- 14 micrograms/ml. The prevalence of fleas with demonstrable cat IgG in their hemolymph 1 h after feeding was 100% for both female and male fleas. Following a single blood meal, cat IgG was present in the hemolymph of all 15 fleas tested 1 h after ingestion but dissipated below detectable levels in 10 of 20 fleas examined 3 h after ingestion, and was detectable in only 1 of 10 fleas examined 18 h after ingestion. However, when fleas were provided with continual access to blood over a 72-h period, IgG content in hemolymph, as measured in excised, triturated legs of individual fleas, remained fairly constant (3-16 pg IgG per sample). Flea feeding studies using specific antisera indicated that IgG in flea hemolymph retained its binding activity, and that at least a portion of the IgG was intact. Passage of ingested host antibody from gut into hemocoel is a prerequisite for the possible development of antiflea vaccines that target antigens outside of the flea midgut lumen (e.g., key components of the flea endocrine system controlling oogenesis).

Facilitation of Rift Valley fever virus transmission by Plasmodium berghei sporozoites in Anopheles stephensi mosquitoes.

Certain mosquito species are susceptible to viral infection but cannot transmit the virus due to a salivary gland barrier. We hypothesized that such species could transmit virus if the mosquito were infected with both virus and malaria parasites. Malaria sporozoites disrupt the integrity of mosquito salivary glands and, in so doing, may destroy salivary gland barriers to viral transmission. To examine this postulate, the model system of Rift Valley fever (RVF) virus and a rodent parasite, Plasmodium berghei, in Anopheles stephensi mosquitoes was used. Viral transmission rates for RVF virus-inoculated anophelines that were previously fed either gametocytemic blood (malaria-infected) or normal blood (control) were compared. Viral transmission rates for anophelines having concurrent sporozoite infection of the salivary glands were 32% (n = 25). None of the RVF virus-inoculated control anophelines (n = 55) transmitted virus. These studies confirm an earlier report that malaria sporozoites can disrupt salivary gland barriers and enhance mosquito transmission of arboviruses. Taken together with similar studies using microfilarial parasites, it is increasingly apparent that mosquito-borne parasites have the potential to enhance mosquito transmission of arboviruses.

Comparison of gene probe and conventional methods for the differentiation of ovine footrot isolates of Dichelobacter nodosus.

In a collaborative study that involved four Australian veterinary diagnostic laboratories a gene probe test based on the recombinant plasmids pJIR318, pJIR314B, and pJIR313, which contain genomic vap or vrl regions, was compared with conventional tests used for the differential diagnosis of ovine footrot. A total of 771 clinical dichelobacter nodosus isolates were tested and designated as belonging to one of several gene probe categories. The results showed that 87% of the virulent isolates belonged to gene probe category 1, compared to only 6% of the benign isolates. It was concluded that there was good correlation between the gene probe test and the virulence designation of these isolates as well as the results of elastase, gelatin-gel and protease isoenzyme tests. Furthermore, the gene probe test was converted to a polymerase chain reaction (PCR)-based test. It is suggested that diagnostic laboratories consider carrying out both this PCR test and tests based on the extracellular proteases of D. nodosus.

Presence of calreticulin in vector fleas (Siphonaptera).

Calreticulin has been defined in the cat flea, Ctenophalides felis (Bouché), and oriental rat flea, Xenopsylla cheopis (Rothschild). Calreticulin, a major endoplasmic reticulum protein, was previously identified as a component of ixodid tick saliva. Using a riboprobe generated from tick calreticulin complementary DNA (cDNA), we distinguished 2 transcripts for calreticulin in cat fleas by Northern blot analysis. Increased expression of calreticulin was not evident in fed versus unfed adult fleas. We were able to amplify a calreticulin flea product from fed female messenger RNa (mRNA) using primers designed from the tick calreticulin gene. One of these products hybridized to the tick riboprobe. Localization of specific antibody to cat flea tissues showed calreticulin in the midgut with no detection in the salivary glands. We also observed specific labeling of calreticulin with antibody in the ovaries of fed females. Several cat flea polypeptides appear to crossreact with anticalreticulin antibody in Western blots. We did not detect a calreticulin using antibody to the tick-secreted protein in cat flea salivary glands. This antibody did recognize a protein in the rate flea salivary glands. Our results show that fleas have calreticulin and, possibly, several isoforms. It appears that the salivary glands of the cat and oriental rat flea differ in detectable levels of calreticulin. The specific antibody labeling of the ovaries is interesting and remains to be understood. Calreticulin's appearance in the midgut suggests a possible source of calreticulin as a flea secretion. Further studies are in progress to complete the sequencing of the flea polymerase chain reaction (PCR) product to compare to tick-secreted calreticulin. Comparisons to other blood-feeding arthropods at the protein and gene level are also being done. We hope to define further the expression of calreticulin in fleas, and in general, blood-feeding arthropods, with respect to its role in feeding and pathogen transmission.

Evidence for phospholipase B activity in Fusobacterium necrophorum cultures and its association with hemolysin/leucocidin activities.

Phospholipase B (PLB) activity was present in Fusobacterium necrophorum cultures and it correlated closely with virulence and co-purified with the hemolysin/leucocidin activities. All three activities were associated with a large molecule or molecular complex (6 x 10(2)-2 x 10(3) kDa) exhibiting varying degrees of aggregation. These were present mainly in the culture medium and to a lesser extent in cell sonicates. The PLB and toxin activities were sensitive to heat, dissociating agents, proteolytic enzymes, prolonged purification regimes, freeze-drying and repeated freeze-thawing. The toxin(s) were stable over a broad range of pH, did not require divalent ions or reducing agents and could be kept for several months as an ammonium sulfate precipitate at 4 degrees C, or stored as a concentrated liquid in the presence of proteolytic inhibitors at - 20 degrees C.

Dual host infections: enhanced infectivity of eastern equine encephalitis virus to Aedes mosquitoes mediated by Brugia microfilariae.

When mosquitoes feed on a vertebrate host that is infected concurrently with virus and microfilariae (mf), both pathogens are ingested. If mf penetrate the mosquito midgut, a small portion of the ingested virus may disseminate directly into the mosquito hemocoel. This phenomenon, termed microfilarial enhancement of arboviral transmission, has the potential to enhance the infectivity of arboviruses to mosquitoes. We investigated whether concurrent ingestion of Brugia mf and eastern equine encephalitis virus would enhance the infectivity and subsequent transmissibility of the virus by Aedes mosquitoes. Trials with Ae. triseriatus and B. pahangi mf indicated that microfilarial enhancement was dose dependent. Both a sufficient number of penetrating mf and a sufficient viremia were required for enhancement to occur. Furthermore, studies with B. malayi and three species of Aedes indicated that under comparable conditions of host viremia and microfilaremia, microfilarial enhancement occurred in some mosquito species (i.e., Ae. aegypti and Ae. taeniorhynchus) but not in others (Ae. triseriatus). We suggest that certain key parameters determine whether dual virus/mf host infections will enhance arboviral infectivity to mosquitoes. These include species differences in the capacity of mf to penetrate the mosquito midgut, the amount of virus passing into the hemocoel during mf penetration, and the innate susceptibility of mosquitoes to hemocoelomically introduced virus.

An experimental ovine foot abscess model using a Fusobacterium necrophorum biotype AB.

An experimental procedure is described for the production of foot abscess in sheep that mimics the natural disease. Lesions were produced by the intradermal inoculation of suspensions of Fusobacterium necrophorum biotype AB containing from 5 x 10(2) to 5 x 10(8) bacteria, into interdigital skin devitalized by freezing with liquid nitrogen. A dose of 5 x 10(5) bacteria induced the development of foot abscess in 3 of 4 and 8 of 8 inoculated feet. It was found that to produce foot abscess in devitalized tissue required between 10(3) and 10(6) fewer bacteria than were necessary to produce similar lesions in healthy tissue.

Noninfectious sporozoites in the salivary glands of a minimally susceptible anopheline mosquito.

In studies to evaluate vector-malaria parasite relationships, we have found that Anopheles albimanus is minimally susceptible to the rodent malaria parasite Plasmodium yoelii. Normally, less than 10% of A. albimanus develop oocyst infections compared to 80-100% for Anopheles stephensi and Anopheles freeborni mosquitoes. Although sporozoites produced in A. albimanus invade the salivary glands, they are not infectious to BALB/c or ICR mice. In 11 experiments with sporozoites from A. albimanus, intravenous inoculations of up to 24,000 sporozoites in individual mice failed to produce host infections. In contrast, inoculation of 300 sporozoites obtained from the salivary glands of A. stephensi and A. freeborni always infected mice. The noninfectious sporozoites from A. albimanus were morphologically similar to the infectious sporozoites from A. stephensi and yielded 4+ circumsporozoite precipitin reactions when incubated with a monoclonal antibody against the circumsporozoite protein of P. yoelii. The presence of noninfectious sporozoites in the salivary glands of A. albimanus suggests that this minimally susceptible vector either possesses a toxic factor that abolishes sporozoite infectiousness or lacks a critical substance needed by the sporozoite to become infectious. Sporozoite infectiousness was neither attenuated by incubation of infectious sporozoites with A. albimanus salivary glands nor restored when noninfectious sporozoites were incubated with A. stephensi salivary glands. These studies provide a starting point for defining the biological basis of sporozoite infectivity.

An immunological factor that affects Anopheles gambiae survival.

High titers of antibodies against Anopheles gambiae midguts were produced in New Zealand rabbits to identify midgut targets for an antimosquito vaccine. The serum from one of 8 rabbits (designated R2B6) killed 71.6% (Abbott's adjusted % mortality) of An. gambiae within 7 days. Mosquitoes ingesting R2B6 serum were unable to absorb their blood meal nutrients, resulting in reduced oviposition and egg hatching rates. Anopheles stephensi and Anopheles arabiensis were also killed when ingesting R2B6 serum but Anopheles freeborni, Anopheles albimanus, and Aedes aegypti were not affected. The mosquitocidal factor was a relatively large molecule (> 100,000 MW) maintained at threshold levels in the sera and killing was complement independent. Mortality, however, was not IgG mediated, as determined by protein A-sepharose fractionation. This surprising finding confounds possibilities of using antibodies against whole mosquito midguts as a step in the development of antimosquito vaccines.

Purification of the extracellular acidic proteases of Dichelobacter nodosus.

Dichelobacter nodosus, a Gram negative obligate anaerobe and causative organism of ovine footrot, secretes a family of extracellular acidic serine proteases with pI's in the range of 5.2 to 5.6, and a basic serine protease with a pI of approximately 9.5. Four acidic proteases (V1, V2, V3 and V5) from virulent and five acidic proteases (B1 to B5) from benign strains of D. nodosus were purified by chromatography on Sephadex G-100 and DEAE-Sepharose CL-6B. Proteases V2, V5 and B5 were found to yield two forms (a and b) on purification which probably arise from limited autolysis of the parent molecule. Amino acid compositions, peptide profiles produced on autolysis and apparent Mr on SDS-PAGE of proteases V1-V3 showed that they were similar to each other and to proteases B1 to B4, and that these proteases were clearly distinct from proteases V5 and B5, which were found to be identical.

Plasmodium falciparum: the population structure of mature gametocyte cultures has little effect on their innate fertility.

In vitro cultured Plasmodium falciparum gametocytes were fed to Anopheles gambiae (G3) mosquitoes to identify parasite population characteristics useful for predicting successful mosquito infections. Parameters were collected from an initial study of 90 infections over a two year period and a second study of 55 infections over 12 weeks. Parasite isolate/clone was identified as the most reliable predictor of gametocyte infectiousness. Parameters such as gametocyte age structure (stage IV:V ratio), exflagellation rate and macrogametocyte maturity were not reliable for predicting infectiousness but were useful for monitoring overall culture maturity. Other variables such as gametocyte density, chronological age of the culture at the time of feed, gametocyte sex ratio, asexual parasitemia, and mixing cultures before mosquito feeding were not predictive. Thus, if a reliable parasite isolate or clone is used, there is no need to measure other characteristics of in vitro gametocyte populations because these will not significantly improve one's ability to predict oocyst infection rates.